OBJECTIVETo investigate the effects of antisense oligonucleotide specific to K-ras point mutation on human pancreatic carcinoma cell PC-2 in vitro.

METHODSHuman pancreatic carcinoma cell PC-2 was transducted with antisense oligonucleotide specific to K-ras point mutation by liposome; the expression of target gene was studied with immunohistochemistry and in situ hybridization. The effect on cell proliferation was studied by artificial count, MTT and mass test.

RESULTSThe expression degree of ras protein and K-ras mRNA transducted with antisense oligonucleotide decreased apparently compared with control group and sense oligonucleotide group 48 h after tansduction. The inhibitory effect on cell proliferation was confirmed by artificial count, MTT and mass test.

CONCLUSIONSAntisense oligonucleotide specific to K-ras point mutation has an apparent inhibitory effect on target gene expression and cell proliferation of human pancreatic carcinoma cell in vitro.

Cell Proliferation ; drug effects ; Genes, ras ; genetics ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Pancreatic Neoplasms ; genetics ; pathology ; Point Mutation ; genetics ; Transfection ; Tumor Cells, Cultured

Antineoplastic Agents ; therapeutic use ; Colorectal Neoplasms ; drug therapy ; genetics ; metabolism ; Genes, ras ; genetics ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Molecular Targeted Therapy ; methods ; Mutation ; Neoplasms ; drug therapy ; genetics ; metabolism ; Pancreatic Neoplasms ; drug therapy ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins p21(ras) ; Receptor, Epidermal Growth Factor ; drug effects ; metabolism ; Signal Transduction ; ras Proteins ; genetics ; metabolism

A molecularly cloned human cellular H-ras (c-H-ras) oncogene(pbc N1 plasmid) was treated with N-acetoxyacetylaminofluorene (AAAF) in vitro and subcloned into E.coli. This was done to identify the mutational changes at specific codons of the gene. Guanine nucleotides were identified as the major AAAF binding site of the DNA adduct formed. Base changes in codons 12 and 61 were determined by the analysis of restriction fragment length polymorphism (RFLP) and site specific oligonucleotide hybridization. RFLP was observed due to the loss of the Hpall recognition site at codon 11 and 12 of AAAF-treated c-H-ras gene. Hybridization of AAAF treated c-H-ras with 32P-labeled oligonucleotide probes for the mutant alleles of codon 61 showed no substitutions at codon 61. From these results, it is assumed that AAAF treatment in vitro caused mutation at codon 12 but not at codon 61 of the c-H-ras oncogene and that codon 12 is the primary target of mutation by AAAF
Acetoxyacetylaminofluorene/*pharmacology ; Chromatography, Thin Layer ; Codon ; *DNA Damage ; Electrophoresis, Agar Gel ; Genes, ras/*drug effects/genetics ; Human ; Mutagenesis, Site-Directed ; Oligonucleotide Probes ; Plasmids/drug effects/genetics ; Polymorphism, Restriction Fragment Length

OBJECTIVETo investigate the role of Ras antisense oligoribonucleotide (ASODN) in multidrug resistance (MDR) of pancreatic carcinoma Pc-2 cells.

METHODSRas and P-gp expression was suppressed by Ras ASODN. Sensitivity of Pc-2 cells to chemotherapy was determined by the MTT assay. MDR-1 mRNA level was detected by fluorogenic probe quantitative reverse transcription polymerase chain (RT-PCR) method. Flow cytometry (FCM) was used to detect the accumulative concentration of adriamycin (ADR) in the cells.

RESULTSRas ASODN significantly inhibited the Ras and P-gp expression (P < 0.05), increased the sensitivity of Pc-2 cells to chemotherapeutic agents (P < 0.05), decreased MDR-1 gene level in Pc-2 cells (P < 0.05), and increased the intracellular intake of ADR in Pc-2 cells (P < 0.05).

CONCLUSIONRas ASODN may enhance the sensitivity of multidrug-resistant pancreatic cancer Pc-2 cells to chemotherapeutic agents by regulating MDR-1 gene level.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Cell Line, Tumor ; Down-Regulation ; Doxorubicin ; metabolism ; Drug Resistance, Multiple ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Genes, MDR ; drug effects ; genetics ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; ras Proteins ; biosynthesis ; genetics